A relationship between iron absorption and dietary calcium content has been observed for many years but the reason for this association has remained unclear. Using isolated small intestinal loops, the absorption of test doses of ferrous radioiron was decreased significantly by calcium chloride in a dose related manner in iron-deficient, normal and iron-loaded rats. In normal rats, this inhibitory effect occurred in duodenum, jejunum and ileum but not stomach or colon. Solubility and chromatographic studies at various pH showed that calcium did not alter the physical or ionic state of iron. An intraluminal effect of calcium upon iron was further excluded by showing a significant diminution of iron absorption in the presence of dietary constituents which enhance both the solubility and absorption of iron. Iron absorption from gut loops was increased in rats conditioned on a high calcium diet by comparison to controls indicating that such diets may diminish iron absorption and produce iron deficiency. Ultrastructural studies using specific ferrocyanide staining for both ferrous and ferric iron in rat intestine demonstrated that an inhibitory effect of calcium on iron absorption occurred at the microvillous membrane. Two proteins have been identified in intestinal mucosa which bind both iron and calcium in vivo and in vitro. The previously described vitamin D-dependent calcium binding protein was shown to bind calcium more avidly than iron whereas a 370,000 molecular weight non-ferritin protein was identified which more avidly binds iron than calcium. These findings suggest that competition of the divalent metal for mucosal absorptive binding sites may be of physiological importance to iron absorption in infants and other individuals consuming a diet with a high calcium content.